Supplementary Text

Whole exome sequencing

Matched diagnostic and remission DNA was available for eight iAMP21 patient samples.

Exomes were captured using 3-5ug of DNA per sample and the Sureselect Human All Exon

50Mb V2 (patient 1) or V4 Kit (patients 3, 5, 7, 9, 21, 43 and 44), as recommended (Agilent

Technologies, Santa Clara, USA). Each library was prepared for sequencing using the

Paired-End Library Preparation Kit (Illumina, San Diego, California, USA). The diagnostic

and remission exomes of patient 1 were each sequenced on two lanes of one flowcell on the

Illumina Genome Analyzer IIX (GAIIX) using 75bp paired-end chemistry (Newcastle

University, Newcastle upon Tyne, UK). The libraries of the remaining samples were

sequenced on the Illumina HiSeq 2000 using 100bp paired-end chemistry (AROS Applied

Biotechnology AS, Aarhus, Denmark). Average coverage was 60-fold (range, 28-fold to

100-fold) per patient sample, calculated using DepthOfCoverage (Genome Analysis Toolkit

(GATK), Broad Institute).

Paired end reads of the diagnostic and remission sample were aligned to UCSC hg19 with

appropriate read groups set using BWA MEM algorithm of Burrows-Wheeler Aligner

(version 0.7.10-r789).1 The resultant SAM alignments were then converted to co-ordinate

sorted and indexed BAM files and de-duplicated using Picard tools (version 1.137)

(http://picard.sourceforge.net/). Standard GATK sample preparation was used to perform

indel realignment and base quality score recalibration on the BAM files (GATK version 3.4).2,

3 Haplotype Caller (HC) (GATK) was used to call variants in the BAM file of each sample

according to the current GATK 3.4 JointGenotyping gVCF workflow. Individual de-duplicated

and indel realigned matched diagnostic/remission BAM files were then merged into one BAM

file which underwent a second round of indel realignment, followed by base quality score

recalibration (using GATK 3.4), before undergoing joint tumour/normal variant calling using

MuTect to identify diagnostic variants.4

Diagnostic-specific variants were annotated using Variant Effect Predictor (VEP).5 Variants

within coding exons or splice site regions were selected for further analysis. The functional

effect of individual mutations was assessed using PolyPhen2 and SIFT, for which the scores

were derived by VEP, and/or Mutation Taster.6-8 COSMIC (Catalogue of Somatic Mutations

in Cancer (COSMIC), version 66) and DBSNP (version 137) were used to characterise the

variants which was outputted from VEP.9, 10 Supplementary Table 4 contains a list of somatic

mutations that were predicted to damage the function of the protein by the majority of

prediction tools, when available for analysis (indels and splice site variations could often not

be assessed using PolyPhen2 or SIFT). DBSNP variants with <1% minor allelic frequency

(MAF) were included.

Code availability

The pipeline and associated scripts are available online through GitHub

(https://github.com/MattBashton/MB-GATK-SGE).

Validation of putative mutations

53% (106/199) of candidate somatic mutations were validated using targeted sequencing

approaches or Sanger sequencing (Supplementary Table 4).

Sanger sequencing validations were performed using WGA DNA of the diagnostic and

remission sample. PCR products were generated using specific primers (Supplementary

Table 3) and standard PCR conditions (AmpliTaq Gold® DNA polymerase, Applied

Biosystems, Paisley, UK). Samples with a visible band by gel electrophoresis were purified

using the QIAquick PCR Purification kit (Qiagen, Manchester, UK) and sent for Sanger

sequencing (DBS Genomics, Durham University, Durham, UK). Sequencing traces were

interpreted using FinchTV (Geospiza, Inc, Seattle, USA) and alignment was performed using

BLAT (UCSC) and BLAST (NCBI).11-13

Targeted sequencing was performed using Haloplex Target Enrichment technology (Agilent

Technologies). The assay was designed to capture regions surrounding the putative

mutation using SureDesign (Agilent Technologies). Libraries were prepared using WGA

DNA of the diagnostic and remission material from the eight iAMP21 patient samples, as

recommended (Agilent Technologies). 16 samples were pooled together at equimolar

concentrations and sequenced on the Illumina HiSeq 2000 using 100bp paired-end

chemistry (Edinburgh Genomics, University of Edinburgh, UK). Data analysis and variant

annotation was performed using the protocol that was established to analyse the whole

exome sequencing libraries (read alignment using BWA, mutation calling following the GATK

Best Practices workflow (HC, MuTect) and VEP to annotate the variants).

100% (106/106) of the putative mutations were validated and demonstrated to be somatic

(i.e. no mutant reads were detected in the remission sample). None of the putative mutations

that were selected for validation were undetected by the assay.

References

1 Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010 Mar 1;26(5):589-95.

2 DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nature genetics. 2011 May;43(5):491-8.

3 Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, Levy-Moonshine A, et al. From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Current protocols in bioinformatics / editoral board, Andreas D Baxevanis [et al]. 2013 Oct 15;11(1110):11 0 1- 0 33.

4 Cibulskis K, Lawrence MS, Carter SL, Sivachenko A, Jaffe D, Sougnez C, et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nature biotechnology. 2013 Mar;31(3):213-9.

5 McLaren W, Pritchard B, Rios D, Chen Y, Flicek P, Cunningham F. Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor. Bioinformatics. 2010 Aug 15;26(16):2069-70.

6 Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nature protocols. 2009;4(7):1073-81.

7 Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nature methods. 2010 Apr;7(4):248-9.

8 Schwarz JM, Cooper DN, Schuelke M, Seelow D. MutationTaster2: mutation prediction for the deep-sequencing age. Nature methods. 2014 Apr;11(4):361-2.

9 Forbes SA, Beare D, Gunasekaran P, Leung K, Bindal N, Boutselakis H, et al. COSMIC: exploring the world's knowledge of somatic mutations in human cancer. Nucleic acids research. 2015 Jan;43(Database issue):D805-11.

10 Sherry ST, Ward MH, Kholodov M, Baker J, Phan L, Smigielski EM, et al. dbSNP: the NCBI database of genetic variation. Nucleic acids research. 2001 Jan 1;29(1):308-11.

11 Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: architecture and applications. BMC bioinformatics. 2009;10:421.

12 Kent WJ. BLAT--the BLAST-like alignment tool. Genome research. 2002 Apr;12(4):656-64.

13 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. Journal of molecular biology. 1990 Oct 5;215(3):403-10.

42 iAMP21-ALL diagnostic samples 7 iAMP21-ALL relapse samples

b) SNP 6.0 array 28 diagnostic samples

2 relapse samples

Screen A) Roche 454 technology Hotspot regions of KRAS, NRAS, and BRAF and all coding exons of FLT3 (24 exons),

PTPN11 (16 exons) and NF1* (57 exons)

Screen B) Illumina MiSeq technology B1) Failed genes/exons in samples from screen A)

B2) Validation of putative mutations from screen A) in diagnostic samples and matched remission (if available) and 2 relapse samples B3) Investigative sequencing of all hotspot mutation exons identified from screen A) in 4 diagnostic and 1 relapse sample**

38 diagnostic 4 relapse

Western blot analysis of pERK expression 2 diagnostic

Western blot analysis of pERK expression 4 positive controls: 3 ALL patient samples and 1 cell line 2 negative controls: 1 ALL patient sample and 1 cell line

4 diagnostic 3 relapse

Cytotoxicity assay using RAS/MEK/ERK inhibitors Xenograft cells from 2 RAS mutant iAMP21-ALL diagnostic patient samples***

2 diagnostic

1 diagnostic

3) The downstream implications of RAS pathway abnormalities

2) Screening for RAS pathway copy number abnormalities (CNAs)

Whole exome sequencing 8 x iAMP21-ALL matched diagnostic and remission samples

1 diagnostic

a) SALSA MLPA P335-IKZF1 kit 38 diagnostic samples

6 relapse samples

1) Screening for RAS pathway mutations

Supplementary Figure 1: RAS pathway investigations in iAMP21-ALL. Whole exome sequencing was carried out on 8 matched diagnostic and remission samples, in which RAS pathway mutations were identified at an incidence of 75% (6/8). (1) iAMP21-ALL samples (n=49, 42 diagnostic and 7 relapse) were subsequently screened for RAS pathway abnormalities. Two mutation screens were carried out: (A) screen A was performed using genomic DNA from 38 diagnostic and 4 relapse samples to investigate the mutational hotspot regions of KRAS (exons 2 and 3), NRAS (exons 2 and 3), and BRAF (exons 11 and 15) and all coding exons of PTPN11, FLT3, and NF1 using Roche 454 sequencing technology. *Apart from patient 1, no other NF1 mutations were identified in an additional 20 samples, thus no further cases were screened. Individual exons were amplified using a 4-primer PCR method; custom-made primers and Access Array Barcode Library primers (Fluidigm Technologies, San Francisco, California, USA) were used to generate individual PCR products using the Access Array System (Fluidigm Technologies) or ABI 2700 Thermocycler (Applied Biosystems, Foster City, California, USA). Amplicons were assessed on the Agilent Bioanalyser (DNA 1000 Assay, Agilent Technologies, Santa Clara, USA) and PCR products were pooled at equimolar concentrations. Data analysis was performed using version 3.4.0 SeqNext (JSI Medical Systems, Kippenheim, Germany) and mutations were identified by manual assessment. The average read coverage achieved for screen A was 418 reads (349-577 per gene). Putative mutations were selected for validation (screen B) if: i) present at a similar variant allele frequency (VAF) (% of mutated reads) and in ≥10 reads in both sequencing directions (forward and reverse) or ii) previously reported in COSMIC (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/) and iii) not in a region of homopolymer sequence. (B) Screen B was performed using whole genome amplified (WGA) DNA and employed Illumina MiSeq sequencing technology to: (B1) repeat any exons or genes that failed to sequence in screen A, (B2) validate the putative mutations identified in patient samples from screen A and (B3) identify mutations in the hotspot regions of NRAS (exons 2 and 3), KRAS (exons 2 and 3), BRAF (exons 11 and 15), FLT3 (exons 5, 14, 15 and 20), and PTPN11 (exons 3, 9, and 13) in an additional 4 diagnostic and 1 relapse iAMP21-ALL patient sample. Matched remission (n=9) and/or relapse (n=3) material was sequenced if sufficient material was available to confirm the somatic or diagnostic-specific nature of the mutations identified in Screen A. The average read coverage achieved for screen B was 35,280 (30,250-48,550 per gene). Mutations were confirmed/validated in screen B if present in ≥500 reads and at a similar VAF (%) to screen A. WGA DNA was representative of the original patient material, the VAF (%) and pattern of RAS mutations was similar for both sample derivatives. ** Genomic DNA of B-other (n=66) and high hyperdiploid (n=48) patient samples were screened for mutations in the hotspot regions of NRAS (exons 2 and 3), KRAS (exons 2 and 3) and FLT3 (exons 4, 14, 15 and 20) using the same targeted sequencing approach. These genes/exons were selected due to their high mutation rate in iAMP21-ALL, to enable a comparison of mutational profiles between the three cytogenetic subgroups. Mutations were identified in the B-other and hyperdiploid patient samples if present in ≥50 reads and at a similar VAF (%) in both sequencing directions (forward and reverse).

(2) Copy number profiling was performed to assess the association between copy number abnormalities (CNAs) and mutations in the RAS pathway genes using (a) SALSA MLPA P335-IKZF1 kit on 38 diagnostic and 6 relapse iAMP21-ALL samples and (b) SNP6.0 analysis in 28 diagnostic and 2 relapse cases. (3) The downstream functional effect of RAS pathway mutations was assessed in two patient samples and 4 positive controls (patient samples and cell lines) by western blot analysis of pERK expression. The cytotoxic effect of selumetinib (MEK1/2 pathway inhibitor) was tested using viable cells from the xenografts of primary cells from two iAMP21-ALL samples with RAS pathway mutations and positive pERK expression. *** Two iAMP21-ALL samples (patients 45 and 46b), in which the xenograft material was screened for RAS mutations, were used as controls in the cytotoxicity assay (Supplementary Figure 6). The results of these studies are reported in the manuscript.

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Supplementary Figure 2: The mutational landscape of iAMP21-ALL. The exome of 8 iAMP21-ALL patient samples harboured 186 nucleotide substitutions and 13 insertions or deletions (indels). (A) Transitions were more common than transversions (0.62:0.32) or indels (0.62: 0.06). (B) Mutations were predominantly composed of C:G>T:A substitutions (60%) with C:G>G:C and C:G>A:T mutations representing a smaller proportion (27%) of substitutions. (C) Most C>T substitutions tended to arise at CpG or TpC sites, a mutational signature that has previously been described in ALL and is associated with DNA methylation, an established process in genome evolution and cancer.1,2 1 Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, et al. Signatures of mutational processes in human cancer. Nature. 2013 Aug 22;500(7463):415-21. 2 Papaemmanuil E, Rapado I, Li Y, Potter NE, Wedge DC, Tubio J, et al. RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia. Nature genetics. 2014 Feb;46(2):116-25.

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iv) Co-occurring mutation rate (% of gene mutations that co-

occurred with other RAS pathway mutations)

KRAS 8 15% 100.0%

NRAS 20 18% 69%

FLT3 9 9% 22%

PTPN11 5 35% 75%

NF1 1 39% 100%

BRAF 1 44% 100%

Supplementary Figure 3: The highly variable nature of RAS pathway mutations in iAMP21-ALL. 44 mutations were identified in components of the RAS pathway (KRAS, NRAS, FLT3, PTPN11, NF1, and BRAF) in 26 patient samples (25 diagnostic and 1 relapse (marked by *). Each RAS pathway gene is colour/pattern-coded as depicted in the respective figure. (A) The variant allele frequency (VAF (% of mutated reads / total reads at the base position)) of each RAS pathway mutation (n=44) identified in the iAMP21-ALL samples can be highly variable, ranging from 2-52%. Each bar represents a single mutation (x-axis) and the corresponding VAF is represented on the y-axis. (B) A tabular summary of the (ii) number of mutations reported for each gene, (iii) average VAF (%) of the mutations per gene and iv) proportion of cases that harboured ≥2 mutations (co-existent mutations) per mutated gene. PTPN11, NF1 and BRAF were often mutated at a higher VAF (%) than other RAS pathway genes and FLT3 mutations were present as single mutations more often than the other RAS pathway genes, however, the significance of these findings could not be assessed due to the small cohort size.

* *

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Supplementary Figure 4: RAS pathway mutations were often present in distinct read populations. Images were taken from SeqNext software (JSI Medical Systems) to demonstrate the subclonal nature of RAS pathway mutations in iAMP21-ALL patient samples 5 (A) and 19 (B). Reference genomic sequence and patient sample genomic sequence are shown. Mutations are highlighted in red text and the number of sequencing reads with a specific gene/exon sequence are depicted. (A) NRAS p.G12S and p.G12V mutations were detected in two individual read populations in patient 19; 2962 reads harboured the G->A substitution at base position 34 and 3316 reads had a G->T nucleotide change at base position 35, resulting in the p.G12S and p.G12V mutations, respectively. These two mutations were therefore present in different read populations and represented individual alleles or subclones, the latter of which would increase the mutational burden of RAS pathway mutations to 45% in this patient sample. Approximately half of the sequencing reads (n=5699) contained the normal sequence for NRAS exon 2. Similar findings were reported for 89% (8/9) of iAMP21-ALL samples, in which multiple mutations were identified within the same exon/amplicon. (B) PTPN11 p.N58S (A->G at base position 173) and p.A72T (G->A at base position 214) were identified in the same read population (4907 reads) in patient 5, demonstrating that both mutations were present within the same allele.

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Supplementary Figure 5: The co-occurring nature of RAS pathway mutations in high hyperdiploid and B-other ALL. (A, B) KRAS, NRAS and FLT3 mutations were investigated in patient samples with high hyperdiploid ALL (n=48) (A) or B-other ALL (n=66) (B). Each sample is labelled on the x-axis and the y-axis defines the variant allele frequency (VAF (%)) of each mutation. The pattern/colour of each bar represents the mutated gene, as depicted by the key. Subclonal mutations of KRAS, NRAS and FLT3 were identified in hyperdiploid and B-other ALL. (C) The mutational profile of RAS pathway genes in ALL subgroups. The average VAF (%) and co-existence nature of KRAS, NRAS and FLT3 mutations in high hyperdiploid (n=81), iAMP21- (n=37) and B-other ALL (n=43) that represent each gene are shown. Co-occurring mutation rate represents the % of gene mutations that co-occurred with another RAS pathway mutation. The incidence of gene mutations in the diagnostic sample of patients with high hyperdiploid (n=48) iAMP21- (n=42 (*not including relapse samples) and B-other (n=66) ALL; the co-existence of other RAS gene mutations in the same sample is not represented in this column. FLT3 mutations tend to arise independently of other RAS pathway gene mutations in iAMP21-ALL (Fisher’s exact test, isolated or co-existent FLT3 mutations in iAMP21 and non-iAMP21-ALL, p=0.14).

Hyperdiploidy iAMP21 B-other

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KRAS 34 71% 18% 74% 8 19% 15% 100.0% 14 21% 10% 50%

NRAS 27 56% 15% 85% 20 48% 18% 69% 23 35% 15% 65%

FLT3 20 42% 16% 55% 9 21% 9% 22% 6 9% 18% 50%

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Supplementary Figure 6: RAS pathway mutations are maintained in 1O, 2O, and 3O xenograft models. (A) Summary of the sequencing data from the NGS targeted sequencing method. Mutations were assessed as discussed in Methods, Targeted Sequencing Approaches, of the main article. Three RAS pathway mutations were identified in the diagnostic material of patient 1 (n=2) and 14 (n=1). Variant allele frequency (VAF) represents the proportion of combined forward and reverse reads that harbour the mutation at the respective base position. Mutations were confirmed in the 1O, 2O, and 3O xenograft material and at similar variant allele frequencies to the diagnostic sample. NRAS (exons 2 and 3), KRAS (exons 2 and 3), FLT3 (exons 14, 15 and 20) and PTPN11 (exons 3, 9 and 13) mutations were not identified in the xenograft material of patients 45 or 46b. These RAS pathway genes/exons were specifically selected for sequencing as they were recurrently mutated in iAMP21 ALL patient samples. Xenograft cells from these patients were used as a control to assess the sensitivity of unmutated cells to selumetinib. (B) Cytotoxicity assays were performed using the MEK1/2 inhibitor, selumetinib, on viable cells from the xenografts of patients 1, 14 , 45 and 46b. Reduced cell viability was observed in response to selumetinib in vitro in patient 1 and 14 only, comparable to levels observed in other RAS mutant ALL samples (red) from our previous study.1 Similarly, the response seen in patients 45 and 46b was equivalent to other non-mutated ALL samples (green). (C) pERK expression was assessed in patient 7 (bold text), relative to three RAS mutant samples. pERK was observed in the positive control samples and absent in patient 7, which harboured FLT3-ITD (p.P606_R607ins10, VAF=4%), demonstrating that this FLT3 mutation did not activate the RAS/RAF/ERK pathway, or at least not to detectable levels . References 1. Irving J, Matheson E, Minto L, Blair H, Case M, Halsey C, et al. Ras pathway mutations are

prevalent in relapsed childhood acute lymphoblastic leukemia and confer sensitivity to MEK inhibition. Blood. 2014 Nov 27;124(23):3420-30.

Supplementary Figure 7: The RAS/RAF/ERK and PI3K/AKT/mTOR pathways – a complex network. FLT3 receptor (blue) dimerisation and anchorage at the cell membrane encourages FLT3 ligand (dark red) binding, which leads to auto-phosphorylation of the receptor (‘P’ hexagon symbol), and activation of the RAS/RAF/MEK (green) and PI3K/AKT/mTOR (red) pathways. Mutant FLT3 receptor can directly activate STAT5, usually regulated by the cytokine receptor/JAK/TYK2 pathway.1 RAS activity is regulated by NF1, and the active state is represented with the yellow explosion symbol. Some genes can co-activate components of both pathways, i.e. RAS can activate RAF and PI3K, and inhibition of one pathway may therefore lead to activation of the other.2 Furthermore, mechanisms of drug resistance can evolve in a sample treated with one form of inhibitor, though the acquisition of mutations in genes involved in the alternative pathway.3 The administration of multiple inhibitors that target a number of key cell-signalling pathways, or in combination with chemotherapeutic agents, may overcome the issue of pathway co-activation or drug resistance. Mutations were detected in FLT3, SHP2 (PTPN11), GRP3 (RASGRP3), RAS, NF1, and BRAF (highlighted by the red lightning bolt) in our iAMP21-ALL cohort. Several components of the RAS/RAF/MEK and PI3K/AKT/mTOR pathways are the current focus of novel targeted therapeutic development; examples of targetable genes (numbered stars) and developed therapies are shown in the white box (http://www.mycancergenome.org/content/other/molecular-medicine/anticancer-agents/). *Therapeutic agents that exhibit multi-gene inhibition. References: 1. Choudhary C, Brandts C, Schwable J, Tickenbrock L, Sargin B, Ueker A, et al. Activation mechanisms of STAT5 by oncogenic Flt3-ITD.

Blood. 2007 Jul 1;110(1):370-4. 2. Steelman LS, Chappell WH, Abrams SL, Kempf RC, Long J, Laidler P, et al. Roles of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR

pathways in controlling growth and sensitivity to therapy-implications for cancer and aging. Aging. 2011 Mar;3(3):192-222. 3. Dail M, Wong J, Lawrence J, O'Connor D, Nakitandwe J, Chen SC, et al. Loss of oncogenic Notch1 with resistance to a PI3K inhibitor in

T-cell leukaemia. Nature. 2014 Sep 25;513(7519):512-6.

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1 23229 437 iAMP21 Diagnosis Y Y Y 454 454 454 454 454 454 Y1b 23229 Remission Y Y Y2 11005 416 iAMP21 Diagnosis Y Y 454 454 454 454 454 454 Y2b 11005 Remission Y Y3 11564 421 iAMP21 Diagnosis Y Y Y 454 454 454 454 454 454 Y3b 11564 Remission Y Y Y4 22129 432 iAMP21 Diagnosis Y Y 454 454 454 454 454 454 Y4b 22129 Remission Y Y5 24207 446 iAMP21 Diagnosis Y Y Y 454 454 454 454 454 454 Y5b 24207 Remission Y Y Y6 2904 451 iAMP21 Diagnosis Y Y 454 454 454 454 454 454 Y6b 2904 iAMP21 Relapse Y Y7 24259 447 iAMP21 Diagnosis Y Y Y 454 454 454 454 454 454 Y7b 24259 Remission Y Y Y8 22007 431 iAMP21 Diagnosis Y Y 454 454 454 454 454 454 Y8b 22007 Remission Y Y9 5898 481 iAMP21 Diagnosis Y Y Y 454 454 454 454 454 454 Y9b 5898 Remission Y Y10 3382 453 iAMP21 Diagnosis Y Y 454 454 454 454 454 454 Y11 22340 434 iAMP21 Diagnosis Y Y 454 454 454 454 454 Y11b 22340 Remission Y Y12 8743 505 iAMP21 Diagnosis Y Y 454 454 454 454 454 Y12b 8743 iAMP21 Relapse Y Y 454 454 454 454 454 Y13 12085 424 iAMP21 Diagnosis Y Y 454 454 454 454 454 Y13b 12085 Remission Y Y14 21567 430 iAMP21 Diagnosis Y Y 454 454 454 IM 454 Y14b 21567 Remission Y15 22322 433 iAMP21 Diagnosis Y Y 454 454 454 454 454 Y15b 22322 Remission Y16 20724 429 iAMP21 Diagnosis Y Y 454 454 454 454 45416b 20724 Remission Y17 6783 486 iAMP21 Diagnosis Y Y 454 454 454 454 454 Y18 9864 512 iAMP21 Diagnosis Y Y 454 454 454 454 454 Y19 3743 456 iAMP21 Diagnosis Y Y 454 454 454 454 454 Y20 5674 477 iAMP21 Diagnosis Y Y 454 454 454 454 454 Y21 23982 444 iAMP21 Diagnosis Y Y 454 454 454 454 454 454 Y21b 23982 Remission Y Y Y22 25452 521 iAMP21 Diagnosis Y IM IM IM IM IM Y23 25453 522 iAMP21 Diagnosis Y IM IM IM IM IM Y24 25454 523 iAMP21 Diagnosis Y IM IM IM IM IM Y24b 25454 iAMP21 Relapse Y IM IM IM IM IM Y25 25455 524 iAMP21 Diagnosis Y IM IM IM IM IM Y26 24655 195 iAMP21 Diagnosis Y 454 454 454 454 45426b 24655 Remission Y27 23765 442 iAMP21 Diagnosis Y 454 454 454 454 454 454 Y27b 23765 Remission Y28 9028 511 iAMP21 Diagnosis Y 454 454 454 454 454 454 Y28b 9028 Remission 454 454 454 454 454 454 Y29 24447 320 iAMP21 Diagnosis Y 454 454 454 454 454 454 Y30 24452 370 iAMP21 Diagnosis Y 454 454 454 IM 454 454 Y31 6008 482 iAMP21 Diagnosis Y 454 454 454 454 454 454 Y32 11061 417 iAMP21 Diagnosis Y 454 454 454 454 454 454 Y33 4134 461 iAMP21 Diagnosis Y 454 454 454 454 454 Y33b 4134 iAMP21 Relapse Y 454 454 454 454 454 454 Y34 4279 465 iAMP21 Diagnosis Y 454 454 454 454 454 Y34b 4279 iAMP21 Relapse Y 454 454 454 454 454 454 Y35 4780 473 iAMP21 Diagnosis Y 454 454 454 IM 454 Y35b 4780 iAMP21 Relapse Y 454 454 454 454 454 45436 4405 467 iAMP21 Diagnosis Y 454 454 454 454 IM Y36b 4405 iAMP21 Relapse Y Y37 7828 502 iAMP21 Diagnosis Y 454 454 454 454 454 Y38 11158 418 iAMP21 Diagnosis Y 454 454 454 454 454 Y39 11557 420 iAMP21 Diagnosis Y 454 454 454 454 454 Y40 6996 491 iAMP21 Diagnosis Y 454 454 454 454 454 Y41 6788 487 iAMP21 Diagnosis Y 454 454 454 IM 454 F42 24454 372 iAMP21 Diagnosis Y 454 454 454 454 45443 23299 439 iAMP21 Diagnosis Y Y Y43b 23299 Remission Y Y44 23888 444 iAMP21 Diagnosis Y Y Y44b 23888 Remission Y Y45* 19578 426 iAMP21 Diagnosis IM IM IM IM46b* 23317 iAMP21 Relapse IM IM IM IM47 8947 B-other Diagnosis Y Y Y48 9146 B-other Diagnosis Y Y Y49 9465 B-other Diagnosis Y Y Y50 9525 B-other Diagnosis Y Y Y51 9859 B-other Diagnosis Y Y Y52 9877 B-other Diagnosis Y Y Y53 10054 B-other Diagnosis Y Y Y54 10077 B-other Diagnosis Y Y Y55 10173 B-other Diagnosis Y Y Y56 10184 B-other Diagnosis Y Y Y57 10186 B-other Diagnosis Y Y Y58 10297 B-other Diagnosis Y Y Y59 10419 B-other Diagnosis Y Y Y60 10442 B-other Diagnosis Y Y Y61 10656 B-other Diagnosis Y Y Y62 10743 B-other Diagnosis Y Y Y63 10817 B-other Diagnosis Y Y Y64 11104 B-other Diagnosis Y Y Y65 11111 B-other Diagnosis Y Y Y66 11390 B-other Diagnosis Y Y Y67 11832 B-other Diagnosis Y Y Y68 11889 B-other Diagnosis Y Y Y69 11957 B-other Diagnosis Y Y Y70 12021 B-other Diagnosis Y Y Y71 12286 B-other Diagnosis Y Y Y72 12324 B-other Diagnosis Y Y Y73 12476 B-other Diagnosis Y Y Y74 12634 B-other Diagnosis Y Y Y75 20323 B-other Diagnosis Y Y Y76 20515 B-other Diagnosis Y Y Y77 20548 B-other Diagnosis Y Y Y78 20574 B-other Diagnosis Y Y Y79 20683 B-other Diagnosis Y Y Y80 20709 B-other Diagnosis Y Y Y81 20716 B-other Diagnosis Y Y Y82 20720 B-other Diagnosis Y Y Y83 20748 B-other Diagnosis Y Y Y84 20750 B-other Diagnosis Y Y Y85 20753 B-other Diagnosis Y Y Y86 20759 B-other Diagnosis Y Y Y87 20764 B-other Diagnosis Y Y Y88 20874 B-other Diagnosis Y Y Y89 21198 B-other Diagnosis Y Y Y90 21207 B-other Diagnosis Y Y Y91 21208 B-other Diagnosis Y Y Y92 22194 B-other Diagnosis Y Y Y93 22387 B-other Diagnosis Y Y Y94 22388 B-other Diagnosis Y Y Y95 22408 B-other Diagnosis Y Y Y96 22499 B-other Diagnosis Y Y Y97 22572 B-other Diagnosis Y Y Y98 22584 B-other Diagnosis Y Y Y99 22673 B-other Diagnosis Y Y Y100 22689 B-other Diagnosis Y Y Y101 22790 B-other Diagnosis Y Y Y102 22861 B-other Diagnosis Y Y Y103 22889 B-other Diagnosis Y Y Y104 22918 B-other Diagnosis Y Y Y105 22936 B-other Diagnosis Y Y Y106 22964 B-other Diagnosis Y Y Y107 22972 B-other Diagnosis Y Y Y108 22980 B-other Diagnosis Y Y Y109 23065 B-other Diagnosis Y Y Y110 23075 B-other Diagnosis Y Y Y111 23114 B-other Diagnosis Y Y Y112 20399 B-other Diagnosis Y Y Y113 8768 HeH Diagnosis Y Y Y114 9154 HeH Diagnosis Y Y Y115 10069 HeH Diagnosis Y Y Y116 10247 HeH Diagnosis Y Y Y117 10254 HeH Diagnosis Y Y Y118 10400 HeH Diagnosis Y Y Y119 10410 HeH Diagnosis Y Y Y

120 10576 HeH Diagnosis Y Y Y121 10626 HeH Diagnosis Y Y Y122 10631 HeH Diagnosis Y Y Y123 10720 HeH Diagnosis Y Y Y124 10740 HeH Diagnosis Y Y Y125 10837 HeH Diagnosis Y Y Y126 10996 HeH Diagnosis Y Y Y127 11103 HeH Diagnosis Y Y Y128 11154 HeH Diagnosis Y Y Y129 11301 HeH Diagnosis Y Y Y130 11407 HeH Diagnosis Y Y Y131 11570 HeH Diagnosis Y Y Y132 11635 HeH Diagnosis Y Y Y133 11674 HeH Diagnosis Y Y Y134 11735 HeH Diagnosis Y Y Y135 11884 HeH Diagnosis Y Y Y136 12325 HeH Diagnosis Y Y Y137 12376 HeH Diagnosis Y Y Y138 12628 HeH Diagnosis Y Y Y139 12728 HeH Diagnosis Y Y Y140 12747 HeH Diagnosis Y Y Y141 19646 HeH Diagnosis Y Y Y142 19764 HeH Diagnosis Y Y Y143 19994 HeH Diagnosis Y Y Y144 20384 HeH Diagnosis Y Y Y145 20712 HeH Diagnosis Y Y Y146 20781 HeH Diagnosis Y Y Y147 21346 HeH Diagnosis Y Y Y148 21635 HeH Diagnosis Y Y Y149 21750 HeH Diagnosis Y Y Y150 21998 HeH Diagnosis Y Y Y151 22147 HeH Diagnosis Y Y Y152 22238 HeH Diagnosis Y Y Y153 22341 HeH Diagnosis Y Y Y154 22420 HeH Diagnosis Y Y Y155 22434 HeH Diagnosis Y Y Y156 22468 HeH Diagnosis Y Y Y157 22676 HeH Diagnosis Y Y Y158 22799 HeH Diagnosis Y Y Y159 22928 HeH Diagnosis Y Y Y160 23031 HeH Diagnosis Y Y Y

References1 Harrison CJ, Moorman AV, Schwab C, Carroll AJ, Raetz EA, Devidas M, et al. An international study of intrachromosomal amplification of chromosome 21 (iAMP21): cytogenetic characterization and outcome. Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, UK. 2014 May;28(5):1015-21.2 Rand V, Parker H, Russell LJ, Schwab C, Ensor H, Irving J, et al. Genomic characterization implicates iAMP21 as a likely primary genetic event in childhood B-cell precursor acute lymphoblastic leukemia. Blood. 2011 Jun 23;117(25):6848-55.

Supplementary Table 1: A summary of genetic studies per patient sample used in this study. Studies were approved by the relevant institutional ethics committee(s) and written informed consent was obtained for each patient from parents, legal guardians or themselves. The cohort was comprised of 46 samples from patients with iAMP21-ALL. Cases 1-44 were derived from patient material which was used to investigate the incidence of RAS mutations in iAMP21-ALL. *Patients 45 and 46b refers to the xenograft material of the patient sample; viable cells were used as a control to assess the effect of selumetinib in iAMP21-ALL (Supplementary Figure 6). Matched remission and /or relapse material was available for select iAMP21-ALL cases (highlighted in grey). Clinical and cytogenetic data is available from our previous studies.1,2 iAMP21-ALL was confirmed in each case through the acquisition of multiple RUNX1 signals (relative to ETV6 ) on interphase cells or metaphase spreads by FISH. 66 (patients 47-112) and 48 (patients 113-160) diagnostic patient samples with no recurrent chromosomal abnormality (defined as B-other) or high hyperdipoidy (HeH), respectively, were used in this study. Genetic studies performed in this study are highlighted in yellow and included SNP6.0 array, MLPA analysis (P335-IKZF1 kit) and targeted sequencing (using Roche 454 or Illumina MiSeq technology). Abbreviations: Y, analysis performed; 454, Roche 454 sequencing technology; IM, Illumina MiSeq sequencing; F, failed.

Pat

ient

ID

Dis

ease

sta

ge

SNP

6.0

arra

y

NR

AS

chr1

:115

2470

84-1

1525

9515

SO

S1

chr2

:392

0868

9-39

3476

04

RA

F1ch

r3:1

2625

099-

1270

5700

BR

AF

chr7

:140

4338

12-1

4062

4564

HR

AS

chr1

1:53

2241

-535

550

CB

Lch

r11:

1190

7698

5-11

9178

859

KR

AS

chr1

2:25

3581

79-2

5403

854

PTP

N11

chr1

2:11

2856

535-

1129

2472

7

FLT3

chr1

3:28

5774

10-2

8674

729

MA

P2K

1ch

r15:

6667

9210

-667

8388

2

MA

PK

3ch

r16:

3012

5425

-301

3463

0

CR

Kch

r17:

1324

646-

1359

561

NF1

chr1

7:29

4219

44-2

9704

695

GR

B2

chr1

7:73

3141

56-7

3401

790

MA

P2K

2ch

r19:

4090

319-

4124

126

MA

PK

1ch

r22:

2212

3318

-222

2197

0

Cop

y nu

mbe

r abn

orm

ality

(C

NA

) inf

orm

atio

n

1 Diagnosis Y 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 BRAF loss, chr7:138489883-141615098, CN=11b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 22 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 MAPK1 gain, chr22, CN=32b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 23 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 23b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 24 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 24b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 25 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 25b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 26 Diagnosis Y 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 BRAF loss, chr7:123075091-159117317, CN=16b Relapse7 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 27b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 28 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 28b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

9 DiagnosisY

2 2 2 2 2 2 2 2 3 2 2 1 3 3 2 2

FLT3 gain chr13:28577688-28653242, CN=3NF1 and GRB2 gain, chr17:21524160-81049726, CN=3CRK loss, chr17:21524160-81049726, CN=1

9b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

10 DiagnosisY

2 2 2 2 2 1 2 2 2 2 2 2 2 3 2 2CBL loss, chr11:101085126-128054642, CN=1GRB2 gain, chr17:40024271-80907559, CN=3

11 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 211b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 212 Diagnosis Y 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 CBL loss, chr11:96439395-134944770, CN=112b Relapse Y 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 CBL loss, chr11:96439395-134944770, CN=113 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 NF1 loss chr17:27671072-30298842, CN=113b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 214 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 3 3 3 2 2 NF1 , GRB2 and CRK gain, chr17, CN=314b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 215 Diagnosis Y 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 CBL loss chr11:103700452-122725504, CN=115b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 216 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 216b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 217 Diagnosis Y 2 2 2 2 2 2 2 2 2 4 2 2 2 2 2 2 MAP2K1 gain chr15:60309674-68683575, CN=418 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 219 Diagnosis Y 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 CBL loss, chr11:78035832-134944770, CN=120 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 221 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 221b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 222 Diagnosis Y 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 FLT3 gain, chr13:21197493-46080684, CN=323 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 224 Diagnosis Y 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 BRAF loss chr7:120150796-159119708, CN=124b Relapse Y 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 BRAF loss chr7:120150796-159119708, CN=125 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 226 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 226b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

43 Diagnosis Y 2 2 2 2 2 1 22

2 3 2 12

2 2 2

CBL loss chr11:89850834-134944770, CN=1MAP2K1 gain chr15:62606091-71815395, CN=3CRK loss chr17:0-16571504, CN=1

43b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 244 Diagnosis Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 244b Remission Y 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Supplementary Table 2: Copy number status of RAS pathway genes in iAMP21-ALL. SNP6.0 copy number profiling was performed on 28 diagnostic and 2 relapse iAMP21-ALL patient samples. Matched remission material was available for 17 cases. Arrays were prepared by The Paterson Institute Microarray Service (Manchester, UK) or AROS Applied Biotechnology AS (Aarhus, Denmark). Copy number and segmentation analysis was performed using Genotyping Console version 4.1.4 (Affymetrix, Santa Clara, California, USA). Regions of genomic gain and loss were confirmed by manual assessment. Matched remission samples facilitated the exclusion of germline copy number variants (CNV) and were used to eliminate potential germline variants in unmatched diagnostic samples, in addition to the list of CNV reported in the Toronto Database of Genomic Variants (DGV). Regions of CNA were mapped in relation to the Genome Reference Consortium Human genome build 37 (GRCh37). The copy number status of 16 genes that represent components of the RAS pathway is shown. Most deletions formed part of a larger abnormality (T) which was often visible by karyotype analysis.

Gene Name Chromosome Exon # Forward primer sequence (+ CS1 - tag) Reverse primer sequence (+ CS2 - tag) Forward primer sequence (+ CS1 - tag) Reverse primer sequence (+ CS2 - tag)KRAS 1 2 ACACTGACGACATGGTTCTACACGTCTGCAGTCAACTGGAAT TACGGTAGCAGAGACTTGGTCTAGAATGGTCCTGCACCAGTAAKRAS 1 3 ACACTGACGACATGGTTCTACATGTAATAATCCAGACTGTGTTTCTCC TACGGTAGCAGAGACTTGGTCTCAGGGATATTACCTACCTCATAAACANRAS 12 2 ACACTGACGACATGGTTCTACAGGCCGATATTAATCCGGTGT TACGGTAGCAGAGACTTGGTCTTCCGACAAGTGAGAGACAGGNRAS 12 3 ACACTGACGACATGGTTCTACACCCTCCCTGCCCCCTTAC TACGGTAGCAGAGACTTGGTCTCAATGTCAAACAACCTAAAACCABRAF 11 11 ACACTGACGACATGGTTCTACATCTTCCTGTATCCCTCTCAGG TACGGTAGCAGAGACTTGGTCTAGTTTATTGATGCGAACAGTGABRAF 11 15 ACACTGACGACATGGTTCTACATTGACTCTAAGAGGAAAGATGAAG TACGGTAGCAGAGACTTGGTCTGTAACTCAGCAGCATCTCAGGPTPN11 12 2 ACACTGACGACATGGTTCTACAGGGACAGGGAAGGTCTTGAT TACGGTAGCAGAGACTTGGTCTAAATGCAGGCAGCAAGCTATPTPN11 12 3 ACACTGACGACATGGTTCTACACGTTCCTTGGGTTTCTTTCA TACGGTAGCAGAGACTTGGTCTCACAAGGGAGCAGCAGACTTPTPN11 12 4 ACACTGACGACATGGTTCTACATTGATCAATCCCTTGGAGGA TACGGTAGCAGAGACTTGGTCTACATCTTGCCAGACCCATTTPTPN11 12 5 ACACTGACGACATGGTTCTACAGAGAGTGCTTGAAAACACTAATGTAA TACGGTAGCAGAGACTTGGTCTTGATAGTTGAAGCTGCAATGGPTPN11 12 6 ACACTGACGACATGGTTCTACACCTCTGTCCGTGCCTTTATG TACGGTAGCAGAGACTTGGTCTGTGTCAATCAATGGCCGAATPTPN11 12 7 ACACTGACGACATGGTTCTACAGCCTGACCCAGATGAACATT TACGGTAGCAGAGACTTGGTCTCCGATGTGCTAACAAGAGCAPTPN11 12 8 ACACTGACGACATGGTTCTACACATCAGGCAGTGTTCACGTT TACGGTAGCAGAGACTTGGTCTCTTTCAGGACATGAGGAAGGAPTPN11 12 9 ACACTGACGACATGGTTCTACATCATCATGGTAAGCTTTGCTTT TACGGTAGCAGAGACTTGGTCTTTCCTAAACATGGCCAATCTGPTPN11 12 10 ACACTGACGACATGGTTCTACAAGATGCGAAACAGGCCATT TACGGTAGCAGAGACTTGGTCTGAGTAACGGCAAGACCCTGAPTPN11 12 11.1 ACACTGACGACATGGTTCTACATAGAACCGGGTGATTCCTCA TACGGTAGCAGAGACTTGGTCTTGCAGTTGCTCTATGCCTCAPTPN11 12 11.2 ACACTGACGACATGGTTCTACACAGCCTGTCCCTGTCTCCTA TACGGTAGCAGAGACTTGGTCTCCCACCACCTCAATACCTACAPTPN11 12 12 ACACTGACGACATGGTTCTACACAGGCACGTTGTACTGGAGA TACGGTAGCAGAGACTTGGTCTCGTGAGCACTTTCCTTCCATPTPN11 12 13 ACACTGACGACATGGTTCTACACTGTAGCCATTGCAACATGC TACGGTAGCAGAGACTTGGTCTCCAAGAGGCCTAGCAAGAGAPTPN11 12 14 ACACTGACGACATGGTTCTACACCCTCACATGTGCACTCTTC TACGGTAGCAGAGACTTGGTCTCCTGGCCAGCTTAGGAAAPTPN11 12 15 ACACTGACGACATGGTTCTACATCTCTCCCTGGGAATGTCAG TACGGTAGCAGAGACTTGGTCTCCAAATTGCTTGCCTGCTTAFLT3 13 2 ACACTGACGACATGGTTCTACATGGTTCACAGGAAGACAGCA TACGGTAGCAGAGACTTGGTCTTGGTAGAGGACTTGCCACAAFLT3 13 3 ACACTGACGACATGGTTCTACATTTGGTCATGCATTTGGAAG TACGGTAGCAGAGACTTGGTCTAGCGTGAACCGTGACTTCTTFLT3 13 4 ACACTGACGACATGGTTCTACAGCACCGAGAGAGCAGAGG TACGGTAGCAGAGACTTGGTCTCCTTTCACGTGAGGAATGTGFLT3 13 5 ACACTGACGACATGGTTCTACAGCAGACTGCTGTGAGGGTTT TACGGTAGCAGAGACTTGGTCTTCCATCACCGGTACCTCCTAFLT3 13 6 ACACTGACGACATGGTTCTACATTTGGCAAAAGTGAAGACTGG TACGGTAGCAGAGACTTGGTCTTGGCTTGAATAACCTTCCCATAFLT3 13 7 ACACTGACGACATGGTTCTACAAAAACTTTTGCATTCATTTCCATT TACGGTAGCAGAGACTTGGTCTAGACTGGCAAGCCCAGTTACFLT3 13 8 ACACTGACGACATGGTTCTACAACGGTGCCTTTAGAAACTGG TACGGTAGCAGAGACTTGGTCTCAGAGAACCAAGCCCTCCTAFLT3 13 9 ACACTGACGACATGGTTCTACACATGCCTGGCTTCTCTCATAA TACGGTAGCAGAGACTTGGTCTGCAAACATCCTCTTTGTCATCAFLT3 13 10 ACACTGACGACATGGTTCTACATGCATCTTTGTTGCTGTCCT TACGGTAGCAGAGACTTGGTCTGTGACCGGCTCCTCAGATAAFLT3 13 11 ACACTGACGACATGGTTCTACAGCAACCTGGATTGAGACTCC TACGGTAGCAGAGACTTGGTCTTCTGCAGAACTGCCTATTCCTFLT3 13 12 ACACTGACGACATGGTTCTACACTCTTGGAAACTCCCATTTGA TACGGTAGCAGAGACTTGGTCTTCTGTTTCATCGCTGAGTGACFLT3 13 13 ACACTGACGACATGGTTCTACATCACACACTGACCCTATACTCTCC TACGGTAGCAGAGACTTGGTCTTTCCAATCTGATGACAGCTCACFLT3 13 14 ACACTGACGACATGGTTCTACAGGATTTCTGCAGCGAGTTCT TACGGTAGCAGAGACTTGGTCTCCCAGCCAGTGAGCTTATTTFLT3 13 15 ACACTGACGACATGGTTCTACATTGTAGTAGCCCTTCTAAGCCATT TACGGTAGCAGAGACTTGGTCTCCAGTGAACTTTAGCAAGTGAGGFLT3 13 16 ACACTGACGACATGGTTCTACAAACTCATAAGTGAATTTGCTTCCA TACGGTAGCAGAGACTTGGTCTTCTTCTCAATACGTTTCCCTATGAFLT3 13 17 ACACTGACGACATGGTTCTACACACATTCCACACTACAGCGACT TACGGTAGCAGAGACTTGGTCTGGCTCACCTGGGAATTAGAAFLT3 13 18 ACACTGACGACATGGTTCTACATCTCAAAGTAGTTGCCCTAGGTTT TACGGTAGCAGAGACTTGGTCTAAAGTCTTTGTTCAGAATGTTCAGGFLT3 13 19 ACACTGACGACATGGTTCTACAGCTAAGAACCGGTCACTGAAA TACGGTAGCAGAGACTTGGTCTTCTGAGCTCTTAGCTGAAACCAFLT3 13 20 ACACTGACGACATGGTTCTACAGCAGCTACCATGGATGTGTC TACGGTAGCAGAGACTTGGTCTGACACTGCAGCCACAGCTAAFLT3 13 21 ACACTGACGACATGGTTCTACACTGGTCAAGCTAACGGGTTC TACGGTAGCAGAGACTTGGTCTGGCACATGACAGAAGCTGTGFLT3 13 22 ACACTGACGACATGGTTCTACATGTCAAGAAGGTATTCCTCCACT TACGGTAGCAGAGACTTGGTCTTCCAGCTTGTGTGGGTGTTFLT3 13 23 ACACTGACGACATGGTTCTACATTTAGCCATGGTAGGCTTCAA TACGGTAGCAGAGACTTGGTCTAGTGGCCAATTTCCTCTGATTFLT3 13 24 ACACTGACGACATGGTTCTACAAGGCAAGTAGTGGGCGAGT TACGGTAGCAGAGACTTGGTCTGCATCCTTAAGAGAGCCACCTNF1 17 2 ACACTGACGACATGGTTCTACATTGGAGGTTGATTGAAAATCG TACGGTAGCAGAGACTTGGTCTAGCAAATTCCCCAAAACACANF1 17 3 ACACTGACGACATGGTTCTACATGCCATTTCTGTTTGCCTTAG TACGGTAGCAGAGACTTGGTCTTTCTCAAGATCAAATTCACAAAGCNF1 17 4 ACACTGACGACATGGTTCTACATCATTATTTTTGTTCTGTGTGTGTG TACGGTAGCAGAGACTTGGTCTTTCATATTACTTCAGTAGTCCCATGTNF1 17 5 ACACTGACGACATGGTTCTACAAGAGATGATGTCTTGCTATGTTGC TACGGTAGCAGAGACTTGGTCTTTCATGATACTAGTTTTTGACCCAGTNF1 17 6 ACACTGACGACATGGTTCTACATGTCCAAGGCATATTTGCTG TACGGTAGCAGAGACTTGGTCTTGATTCAGGATGCTAACAACAGNF1 17 7 ACACTGACGACATGGTTCTACAGCCTGGAAAAGGTAAGTTACAA TACGGTAGCAGAGACTTGGTCTAAAAAAAAAAAATCAATCGTATCCNF1 17 8 ACACTGACGACATGGTTCTACATTTTAAAAATGTTGCCCTTGG TACGGTAGCAGAGACTTGGTCTAAGCCTAAAGTAATACACACCTTGANF1 17 9 ACACTGACGACATGGTTCTACAGCTACATCTGGAATAGAAGAAACTTCATACGGTAGCAGAGACTTGGTCTCGAAGAGTCAGAACTTTAATGTTAGCNF1 17 10 ACACTGACGACATGGTTCTACATGCCATTTGTGTGGGTAATG TACGGTAGCAGAGACTTGGTCTACGAACATCAATACATAATAGTGAGANF1 17 11 ACACTGACGACATGGTTCTACAAATCTGCTTTTTTTTTTCTTTTTC TACGGTAGCAGAGACTTGGTCTTTCCAAAGCATCTTATTAACTCCANF1 17 12 ACACTGACGACATGGTTCTACAGACGTTGTTTGAAGACTTTGGA TACGGTAGCAGAGACTTGGTCTTGCAATAGAAAGGAGGTGAGANF1 17 13 ACACTGACGACATGGTTCTACAACCTTATGCTTACTATTGAGTGTTTCT TACGGTAGCAGAGACTTGGTCTCGTTTCAGCTAAACCCAATTAACNF1 17 14 ACACTGACGACATGGTTCTACATCACCCCTAAAAGAAACTTGG TACGGTAGCAGAGACTTGGTCTTTTCCAGAAATGACATCTACCTGNF1 17 15 ACACTGACGACATGGTTCTACATGAAACTACAAATGAAAGAGCTCAA TACGGTAGCAGAGACTTGGTCTAATACAAAACCATAAAACCTTTGGANF1 17 16 ACACTGACGACATGGTTCTACATGCATTAGGTTATTGATGATGC TACGGTAGCAGAGACTTGGTCTTTGCCAGTCATTGTCCTCTGNF1 17 17 ACACTGACGACATGGTTCTACACTTGGTTGTCAGTGCTTCAG TACGGTAGCAGAGACTTGGTCTCCACAAAGAGCAGTAATAGAACTAGANF1 17 18 ACACTGACGACATGGTTCTACATTTATACATAAAATTACCCAAGTTGC TACGGTAGCAGAGACTTGGTCTTGTGCTTTGAGGCAGACTGANF1 17 19 ACACTGACGACATGGTTCTACATCCTGTGAGGTTAGTGAAAGGAA TACGGTAGCAGAGACTTGGTCTTGCACGTATCTTGGATTTACTTCNF1 17 20 ACACTGACGACATGGTTCTACATGTGATCAGGAATAGCTTTTGA TACGGTAGCAGAGACTTGGTCTTGTTTACTTTACTGAGCGACTCTTGNF1 17 21.1 ACACTGACGACATGGTTCTACATGTCATGGAAGAAATGTTGGA TACGGTAGCAGAGACTTGGTCTCTGCGTTTCCCTCTGAAGACNF1 17 21.2 ACACTGACGACATGGTTCTACAGTGTGCCTCCAGCAGAGAA TACGGTAGCAGAGACTTGGTCTTTCATAGAGAAAGGTGAAAAATAAGANF1 17 22 ACACTGACGACATGGTTCTACATGGGTGCACTTACTCTGTGTG TACGGTAGCAGAGACTTGGTCTAAGAATGGCCAGTTATTTTATCAATNF1 17 23 ACACTGACGACATGGTTCTACATGCCTTCTCTTTTGTCTATATCTGA TACGGTAGCAGAGACTTGGTCTAGGCACAAAGCTTAGGGAAANF1 17 24 ACACTGACGACATGGTTCTACACGTCATGTCACTTAGGTTATCTGG TACGGTAGCAGAGACTTGGTCTGCATCTTTACATATTTCAAACACAAANF1 17 25 ACACTGACGACATGGTTCTACATGAGGGGAAGTGAAAGAACTTG TACGGTAGCAGAGACTTGGTCTGCAAAATTTCACTGATCATATTACTTNF1 17 26 ACACTGACGACATGGTTCTACACCATTCACACCATGCACATA TACGGTAGCAGAGACTTGGTCTTGCTTCTCTTACATGCCAGTTCNF1 17 27 ACACTGACGACATGGTTCTACACTTCAGCAAGGCCATGTTAGT TACGGTAGCAGAGACTTGGTCTTATTCTGAAGGATTTGCTATGTGCNF1 17 28 ACACTGACGACATGGTTCTACATATTAATCAGTCATCATTTGCCTTA TACGGTAGCAGAGACTTGGTCTCCTCAGCACACACATACACACANF1 17 29 ACACTGACGACATGGTTCTACAGACTCTCTTCCGAGGCAACA TACGGTAGCAGAGACTTGGTCTAAACAGCGGTTCTATGTGAAANF1 17 30 ACACTGACGACATGGTTCTACAACGTTGCACTTGGCTTAATG TACGGTAGCAGAGACTTGGTCTCCACACACCATCAGCAGCTANF1 17 31 ACACTGACGACATGGTTCTACATGCTGTATGTAGTCGGTGCTG TACGGTAGCAGAGACTTGGTCTAACAAATGCCCACAAATTGCNF1 17 32 ACACTGACGACATGGTTCTACATGTCATTCATGAGGACTGATTG TACGGTAGCAGAGACTTGGTCTAAAGCTGAAAATTTAGTTGGAAGGNF1 17 33 ACACTGACGACATGGTTCTACATTGGGAAGGTTAGAAACACTACCT TACGGTAGCAGAGACTTGGTCTTGGATTTATGTGAAACCGAAANF1 17 34 ACACTGACGACATGGTTCTACACAAGCCCTCCATATTTGTAATCT TACGGTAGCAGAGACTTGGTCTAAAAGCACTATTCATGACCAATAANF1 17 35 ACACTGACGACATGGTTCTACATGGTCCTGAGGTCTTTTTGG TACGGTAGCAGAGACTTGGTCTTGGTGGCAAACTCTCCTTCTNF1 17 36 ACACTGACGACATGGTTCTACACTCAGTAGACAACATAAAGCCTCA TACGGTAGCAGAGACTTGGTCTGCAGCTACTAGATACCGACCATCNF1 17 37.1 ACACTGACGACATGGTTCTACAAAAATAAAATTGATTAGTGGCATCTG TACGGTAGCAGAGACTTGGTCTAGCCGCTCATGATACTTGGT ACACTGACGACATGGTTCTACACATACCGGGCCTAGCAATC TACGGTAGCAGAGACTTGGTCTGTGTACTCCCTGACCCAGGANF1 17 37.2 ACACTGACGACATGGTTCTACATTCCTGGCTTTGCTTACGAC TACGGTAGCAGAGACTTGGTCTCACCTAGGGAGGCCAGGATANF1 17 38.1 ACACTGACGACATGGTTCTACATCCACTTCACCCCGTCAC TACGGTAGCAGAGACTTGGTCTTGCGATATTGAGCAGTGTCCNF1 17 38.2 ACACTGACGACATGGTTCTACATCACCTTAACCATTGCAAACC TACGGTAGCAGAGACTTGGTCTCCAACACTGCATACCTTCCANF1 17 39 ACACTGACGACATGGTTCTACATGTAACAGAATCACAAATTGTATGTTA TACGGTAGCAGAGACTTGGTCTAAAGGGTTTTCTTTGAATTCTCTTANF1 17 40 ACACTGACGACATGGTTCTACATTGATGTGATTTTCATTGACCA TACGGTAGCAGAGACTTGGTCTGTGTCTAGCGCAGTGCTTTGNF1 17 41 ACACTGACGACATGGTTCTACATTGATTAGGCTGTTCCAATGAA TACGGTAGCAGAGACTTGGTCTGGGACTCAAAGTTTTAGCACAANF1 17 42 ACACTGACGACATGGTTCTACAAAAAAAAAAAATCCTGCTTCTTTAC TACGGTAGCAGAGACTTGGTCTTGGTAAACACAAATTCCTTTCCNF1 17 43 ACACTGACGACATGGTTCTACAATTTTCTGTCTTTACTTGTTCCTTTA TACGGTAGCAGAGACTTGGTCTCAAAATAGCACAATAAACCAATATTTCNF1 17 44 ACACTGACGACATGGTTCTACATGCATGGACTGTGTTATTGG TACGGTAGCAGAGACTTGGTCTTGCAGGGATGGATTATATTGGNF1 17 45 ACACTGACGACATGGTTCTACAATGCATATTGTTGAAAATACAGCTA TACGGTAGCAGAGACTTGGTCTATATTTCATTGACCTCAAATTTAAACGNF1 17 46 ACACTGACGACATGGTTCTACATCCTGAATTCATTCCGAGATTC TACGGTAGCAGAGACTTGGTCTACTTCAAACAACTAAACTTTAAGAGGNF1 17 47 ACACTGACGACATGGTTCTACACCCCAAAAGAGAAAACATGG TACGGTAGCAGAGACTTGGTCTGCAACAAGAAAAGATGGAAGAGNF1 17 48 ACACTGACGACATGGTTCTACATGTTCTGTGGTTTTCTGCAGTC TACGGTAGCAGAGACTTGGTCTTCACTTATTCAAATTACTTCTGGTTTCNF1 17 49 ACACTGACGACATGGTTCTACACCTCAGCAGATGCTTGTTCA TACGGTAGCAGAGACTTGGTCTCCACCACTAAAGGACTAGACTGTGNF1 17 50 ACACTGACGACATGGTTCTACATTCATCCTGTTTTAAGTCACACTTG TACGGTAGCAGAGACTTGGTCTTCACTTACTCTTCCTAGGCCATCNF1 17 51 ACACTGACGACATGGTTCTACAAGGAAATAGGACAGCCACTTG TACGGTAGCAGAGACTTGGTCTCATGGAAAATTTTGATAATCCTGANF1 17 52 ACACTGACGACATGGTTCTACAGATGGAAAATAAAGGAAAGAAACTG TACGGTAGCAGAGACTTGGTCTGACTTTCATGTACTCTCCCACCTNF1 17 53 ACACTGACGACATGGTTCTACAGGTGTTTGATCACGTTAATTCC TACGGTAGCAGAGACTTGGTCTGTAGCCTGCCAAGATGCAANF1 17 54 ACACTGACGACATGGTTCTACAGTCTTCTACTTCTCACCCAAACAGA TACGGTAGCAGAGACTTGGTCTAACCCTCATTAAACCTACTGTCTCANF1 17 55 ACACTGACGACATGGTTCTACAATCTCCCTTTAATTTTGGCACA TACGGTAGCAGAGACTTGGTCTAGTCAGTGCATTCTACAACAGCNF1 17 56 ACACTGACGACATGGTTCTACAACTGTATGTCCAATGTAACTGGTTG TACGGTAGCAGAGACTTGGTCTGTGTGTTCTTAAAGCAGGCATANF1 17 57 ACACTGACGACATGGTTCTACATTAATATTTTTGGCTTCAGATGG TACGGTAGCAGAGACTTGGTCTGTTGGTGTCTTATATTGTTGCTCNF1 17 58 ACACTGACGACATGGTTCTACAAGCGACACATGACTGCAATG TACGGTAGCAGAGACTTGGTCTCAAACCGGATGGGTTCATTA

Gene Name Chromosome Exon #FLT3 13 13 TCCCTTTCATCCAAGACAACAFLT3 13 16 TGAGTGCCTCTCTTTCAGAGC

Supplementary Table 3: Primer sequences used to screen and validate RAS pathway mutations. A) Primers were used to screen for RAS pathway mutations using the Roche 454 FLX and / or Illumina MiSeq. RAS pathway primers were composed of the consensus sequence (red text) for binding of the Access Array Barcode Library primers and the target-specific sequence (black text). NRAS , KRAS , BRAF , and NF1 primers have been described previously.1-3 Primers targeting NRAS exon 2 and FLT3 exon 16 were used to validate putative mutations identified by whole exome sequencing using Sanger sequencing. B) Primers used to amplify FLT3 transcripts that harbour ITD mutations, designed to bind to exons 13 (forward primer) and 16 (reverse primer). CS1 - tag, consensus sequence 1; CS2 - tag, consensus sequence 2.

A) Sequencing Primers for RAS Pathway Analysis Illumina MiSeq Sequencing Primers for RAS Pathway Analysis (if different)

References1 Grossmann V, Kohlmann A, Zenger M, Schindela S, Eder C, Weissmann S, et al. A deep-sequencing study of chronic myeloid leukemia patients in blast crisis (BC-CML) detects mutations in 76.9% of cases. Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, UK. 2011 Mar;25(3):557-60.2 Haferlach C, Grossmann V, Kohlmann A, Schindela S, Kern W, Schnittger S, et al. Deletion of the tumor-suppressor gene NF1 occurs in 5% of myeloid malignancies and is accompanied by a mutation in the remaining allele in half of the cases. Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, UK. 2012 Apr;26(4):834-9.3 Grossmann V, Bacher U, Artusi V, Kohlmann A, Nadarajah N, Kern W, et al. Molecular analysis of RAS-RAF tyrosine-kinase signaling pathway alterations in patients with plasma cell myeloma. Blood cancer journal. 2012;2:e85.

B) RT-PCR Primers to amplify FLT3 ITD

Reference allele

Alternative allele DBSNP site Diagnosis

VAF (%)Remission VAF (%)

Amino acid position

Reference amino acid

Mutated amino acid Type of mutation

1 chr1 17085006 MSTP9 11/15 C T DBSNP 8 3 490 G E Missense Probably damaging -1 chr1 215345428 KCNK2 5/7 C T NOVEL 60 0 227 A V Missense Possibly damaging Disease-causing mutation Yes1 chr1 237586495 RYR2 12/105 G A NOVEL 41 0 318 D N Missense Benign Disease-causing mutation Yes1 chr2 33768636 RASGRP3 12/17 G A NOVEL 39 0 445 E K Missense Probably damaging Disease-causing mutation Yes1 chr2 77746691 LRRTM4 3/4 C T NOVEL 46 0 102 E K Missense Benign Disease-causing mutation Yes1 chr3 33558652 CLASP2 34/38 G A NOVEL 51 0 1267 H Y Missense Possibly damaging Disease-causing mutation Yes1 chr3 44929267 TGM4 3/14 C T NOVEL 50 0 94 Q * Frameshift Probably damaging Disease-causing mutation Yes1 chr6 5613493 FARS2 6/7 G A NOVEL 11 0 386 R Q Missense Probably damaging Disease-causing mutation1 chr9 125054104 MRRF 5/7 G A NOVEL 50 0 179 R Q Missense Possibly damaging Disease-causing mutation1 chr12 6697096 CHD4 24/40 C G COSMIC 44 0 1162 R P Missense Probably damaging Disease-causing mutation Yes1 chr12 22059189 ABCC9 10/38 C T NOVEL 48 0 497 E K Missense Probably damaging Disease-causing mutation Yes1 chr12 26808679 ITPR2 20/57 G A NOVEL 41 0 851 P S Missense Probably damaging Disease-causing mutation1 chr12 55641421 OR6C74 1/1 C T NOVEL 43 0 117 S F Missense Probably damaging Disease-causing mutation Yes1 chr12 62778031 USP15 10/21 G A NOVEL 10 0 445 R H Missense Probably damaging Disease-causing mutation1 chr12 79837985 SYT1 9/10 A C NOVEL 43 0 354 Q P Missense Possibly damaging Disease-causing mutation Yes1 chr12 85257259-85257260 SLC6A15 11/12 CC TT NOVEL 47 0 593 G R Missense Possibly damaging Disease-causing mutation Yes1 chr15 48784689 FBN1 24/66 C T NOVEL 47 0 941 M I Missense Benign Disease-causing mutation Yes1 chr16 784854 NARFL 5/11 C T NOVEL 59 0 153 D N Missense Probably damaging Disease-causing mutation Yes1 chr16 20998752 DNAH3 47/62 G A NOVEL 10 0 2301 R W Missense Probably damaging Disease-causing mutation1 chr16 24580488 RBBP6 17/18 G A COSMIC 9 0 826 R H Missense Probably damaging Disease-causing mutation1 chr17 29653001 NF1 37/58 C T NOVEL 35 0 1667 P S Missense Probably damaging Disease-causing mutation Yes1 chr17 45234397 CDC27 7/19 G A DBSNP 13 0 242 P S Missense Benign Disease-causing mutation1 chr18 19031010 GREB1L 13/33 G A NOVEL 39 0 583 E K Missense Probably damaging Disease-causing mutation Yes1 chr20 32996513 ITCH 4/25 G A NOVEL 67 0 43 V I Missense Probably damaging Disease-causing mutation Yes1 chr20 55803435 BMP7 2/7 C T COSMIC 47 0 154 R Q Missense Benign Disease-causing mutation Yes1 chrX 102977125 GLRA4 6/9 G A DBSNP 58 0 225 R W Missense Probably damaging Benign Yes1 chrX 130409464 IGSF1 16/20 G T NOVEL 28 0 1058 L I Missense Probably damaging Disease-causing mutation

3 chr1 162824712 C1orf110 4/4 G C DBSNP 4 0 251 S * Missense Probably damaging Disease-causing mutation3 chr1 240370120 FMN2 5/18 G C NOVEL 33 0 670 E Q Missense Probably damaging Disease-causing mutation Yes3 chr2 15607931 NBAS - G A NOVEL 22 0 Splice site Disease-causing mutation3 chr2 37121091 STRN 7/18 C G NOVEL 25 0 294 S * Truncation Disease-causing mutation Yes3 chr2 99013327 CNGA3 8/8 C T DBSNP, COSMIC 41 0 565 T M Missense Probably damaging Disease-causing mutation Deleterious3 chr2 167136968 SCN9A 14/27 C T NOVEL 8 0 737 L F Missense Probably damaging Disease-causing mutation Deleterious3 chr2 171811243 GORASP2 6/10 G A NOVEL 31 0 217 G E Missense Benign Disease-causing mutation Deleterious3 chr3 46063244 XCR1 2/2 A C NOVEL 49 0 66 T P Missense Probably damaging Disease-causing mutation Deleterious Yes3 chr3 88205168 C3orf38 - C G NOVEL 26 0 Splice site Disease-causing mutation3 chr4 89579597 HERC3 10/26 C G NOVEL 22 0 367 I M Missense Possibly damaging Disease-causing mutation Deleterious Yes3 chr4 187535486 FAT1 12/27 G A NOVEL 15 0 3030 D N Missense Benign Disease-causing mutation3 chr4 187630726 FAT1 2/27 G A NOVEL 15 0 86 E K Missense Probably damaging Disease-causing mutation Yes3 chr5 111576501 EPB41L4A 10/23 G A DBSNP, COSMIC 45 0 268 E K Missense Benign Disease-causing mutation Deleterious3 chr5 140746136 PCDHGA5 1/4 C T COSMIC 26 0 747 R W Missense Possibly damaging Disease-causing mutation Deleterious3 chr7 40127785 CDK13 12/14 G C NOVEL 15 0 1030 Q H Missense Probably damaging Disease-causing mutation Deleterious3 chr10 48371397 ZNF488 2/2 G A DBSNP 36 0 289 D N Missense Probably damaging Disease-causing mutation Deleterious3 chr10 105377003 SH3PXD2A 10/14 G A NOVEL 37 0 263 V I Missense Possibly damaging Disease-causing mutation Deleterious Yes3 chr11 63971536 STIP1 14/14 A G NOVEL 33 0 524 N D Splice site Probably damaging Disease-causing mutation Deleterious Yes3 chr11 76796027 CAPN5 2/13 T C COSMIC 15 0 32 F S Missense Possibly damaging Disease-causing mutation Deleterious3 chr12 20885882 SLCO1C1 10/15 C T NOVEL 40 0 409 S F Missense Possibly damaging Disease-causing mutation Tolerated Yes3 chr12 26221734 RASSF8 5/5 G A NOVEL 25 0 415 E K Missense Probably damaging Disease-causing mutation Deleterious3 chr13 32813860 FRY 46/61 G A NOVEL 17 0 2177 E K Missense Probably damaging Disease-causing mutation Deleterious Yes3 chr13 33333821 PDS5B 29/35 C T NOVEL 20 0 1122 P L Missense Possibly damaging Disease-causing mutation Tolerated Yes3 chr14 20404065 OR4K1 1/1 G C NOVEL 27 0 80 K N Missense Probably damaging Benign Deleterious Yes3 chr14 54418601 BMP4 4/4 G T NOVEL 26 0 114 A S Missense Probably damaging Disease-causing mutation Deleterious3 chr17 36830270 C17orf96 1/1 C T NOVEL 21 0 160 P L Missense Probably damaging Benign Deleterious Yes3 chr19 34934777 UBA2 7/17 C T NOVEL 39 0 204 Q * Truncation Disease-causing mutation Yes3 chr19 49982165 FLT3LG 1/68 G GC COSMIC 16 0 114-115 Q X Frameshift Disease-causing mutation3 chr19 58151289 ZNF211 4/15 C T NOVEL 22 0 89 S L Missense Possibly damaging Benign Deleterious3 chrX 17768017 SCML1 6/8 C T NOVEL 43 0 103 R * Truncation Disease-causing mutation Yes3 chrX 107898629 COL4A5 37/53 G T NOVEL 50 0 1105 L F Missense Benign Disease-causing mutation Yes3 chrX 117707822 DOCK11 12/53 G C NOVEL 19 0 410 K N Missense Possibly damaging Disease-causing mutation Deleterious Yes

5 chr1 1275023 DVL1 10/15 C G NOVEL 28 0 331 I M Missense Benign Disease-causing mutation Deleterious Yes5 chr1 153782716 GATAD2B 11/11 A ACCCC NOVEL 52 0 573 R RGX Frameshift Disease-causing mutation5 chr1 171759675 METTL13 5/8 G C NOVEL 47 0 465 D H Missense Probably damaging Disease-causing mutation Deleterious Yes5 chr1 173493929 SLC9C2 20/28 G C NOVEL 11 0 835 E Q Missense Possibly damaging Benign Deleterious5 chr1 186294919 TPR 42/51 G A NOVEL 31 0 2030 R K Missense Benign Disease-causing mutation Yes5 chr1 208227783 PLXNA2 14/32 C G NOVEL 42 0 947 Q E Missense Possibly damaging Disease-causing mutation Tolerated Yes5 chr2 170033060 LRP2 54/79 C T DBSNP 10 0 3478 H Y Missense Probably damaging Disease-causing mutation5 chr2 179474056 TTN 273/363 C A NOVEL 9 0 17327 S R Missense Benign Disease-causing mutation5 chr3 47161887-47161891 SETD2 3/21 ACTCT A NOVEL 52 0 1412-1413 ES X Frameshift Disease-causing mutation5 chr3 52089905 DUSP7 1/3 A G NOVEL 43 0 160 K E Missense Benign Disease-causing mutation Deleterious Yes5 chr3 99895209 CMSS1 9/10 G A NOVEL 35 0 236 D N Missense Probably damaging Disease-causing mutation Deleterious5 chr3 110863745 PVRL3 - G A NOVEL 41 0 Splice site Disease-causing mutation5 chr3 113375194 KIAA2018 7/7 C T NOVEL 52 0 1779 Q * Splice site Disease-causing mutation5 chr4 17841343 NCAPG 17/21 G C NOVEL 14 0 837 E D Missense Probably damaging Disease-causing mutation Deleterious5 chr4 57876666 POLR2B 12/26 C T NOVEL 44 0 515 P L Missense Probably damaging Disease-causing mutation Deleterious Yes5 chr4 76733470 USO1 24/25 C T NOVEL 8 0 923 A V Missense Probably damaging Disease-causing mutation5 chr4 123095753 KIAA1109 5/86 C G NOVEL 18 0 80 S C Missense Probably damaging Disease-causing mutation Yes5 chr4 177142616 ASB5 4/7 G A NOVEL 6 0 174 E K Truncation Probably damaging Disease-causing mutation Deleterious5 chr5 173383124 CPEB4 10/10 C A NOVEL 30 0 725 S * Splice site Disease-causing mutation Yes5 chr6 144081539 PHACTR2 5/13 G A NOVEL 38 0 152 Splice site Disease-causing mutation5 chr7 11500386 THSD7A 11/28 G C COSMIC 6 0 836 K N Missense Probably damaging Disease-causing mutation Deleterious5 chr7 138204006 TRIM24 4/19 G A NOVEL 55 0 235 C Y Missense Probably damaging Disease-causing mutation Deleterious Yes5 chr7 138235907 TRIM24 8/19 C T NOVEL 33 0 415 Q * Splice site Disease-causing mutation Yes5 chr7 138749702 ZC3HAV1 8/13 A T COSMIC 67 0 639 E V Missense Benign Disease-causing mutation Deleterious Yes5 chr8 76465357 HNF4G 5/10 G C COSMIC 50 0 180 M I Missense Possibly damaging Disease-causing mutation Tolerated Yes5 chr9 79635850 FOXB2 1/1 C T COSMIC 11 0 427 S L Missense Probably damaging Benign Deleterious5 chr9 125377069 OR1Q1 1/1 C G NOVEL 11 0 18 S C Missense Probably damaging Benign Deleterious5 chr9 130885372 PTGES2 5/7 T G NOVEL 18 0 243 V G Missense Probably damaging Disease-causing mutation Deleterious5 chr10 64952712 JMJD1C 16/26 G C NOVEL 45 0 2021 R T Missense Probably damaging Disease-causing mutation Deleterious Yes5 chr10 90697935 ACTA2 8/9 C G NOVEL 13 0 291 I M Missense Probably damaging Disease-causing mutation Deleterious5 chr11 17741913 MYOD1 1/3 G A NOVEL 7 0 195 G D Missense Probably damaging Disease-causing mutation Deleterious5 chr11 57428405 CLP1 3/3 C G NOVEL 52 0 259 Q E Missense Possibly damaging Disease-causing mutation Deleterious Yes5 chr11 64822077 NAALADL1 5/18 G A COSMIC 33 0 246 R Q Missense Probably damaging Disease-causing mutation Deleterious Yes5 chr11 72632974 FCHSD2 9/20 G T NOVEL 28 0 243 D Y Missense Possibly damaging Disease-causing mutation Deleterious Yes5 chr11 76796027 CAPN5 2/13 T C COSMIC 15 0 32 F S Missense Possibly damaging Disease-causing mutation Deleterious5 chr11 94533290 AMOTL1 3/13 G C NOVEL 30 0 312 E Q Missense Probably damaging Disease-causing mutation Tolerated Yes5 chr11 119103299 CBL 2/16 G C NOVEL 21 0 113 E Q Missense Possibly damaging Disease-causing mutation Deleterious Yes5 chr12 29689147 TMTC1 11/18 G C NOVEL 8 0 594 E Q Missense Probably damaging Disease-causing mutation Tolerated5 chr12 54912519 NCKAP1L - A G NOVEL 5 0 Splice site Disease-causing mutation5 chr12 75785038 GLIPR1L2 1/6 G A COSMIC 20 0 48 E K Missense Probably damaging Disease-causing mutation Tolerated5 chr12 80014945 PAWR 3/7 C T DBSNP 10 0 187 R W Missense Probably damaging Disease-causing mutation Deleterious5 chr12 112888198 PTPN11 3/16 G A DBSNP 67 0 72 A T Missense Probably damaging Disease-causing mutation Deleterious Yes5 chr12 124366181 DNAH10 50/78 G A NOVEL 32 0 2764 E K Missense Possibly damaging Disease-causing mutation Yes5 chr15 52017212 LYSMD2 2/3 G C NOVEL 10 0 127 G A Missense Probably damaging Benign Deleterious5 chr17 15641610 TBC1D26 7/15 A G DBSNP 20 0 99 Y C Missense Possibly damaging Benign Deleterious5 chr17 78064000-78064064 CCDC40 18/18 AGGCACG A NOVEL 67 0 966-987 GTCTKNTG X Frameshift Disease-causing mutation5 chr19 38103007 ZNF540 5/5 T C NOVEL 50 0 276 C R Missense Probably damaging Disease-causing mutation Deleterious Yes5 chrX 70344126 MED12 13/45 G A NOVEL 17 0 621 R Q Missense Probably damaging Disease-causing mutation Yes5 chrX 109694000 RGAG1 3/4 C T NOVEL 7 0 52 S L Missense Possibly damaging Benign Deleterious5 chrX 117119202 KLHL13 - C A NOVEL 38 0 Splice site Disease-causing mutation5 chrX 154743751 TMLHE 4/8 G C NOVEL 48 0 178 K N Missense Probably damaging Disease-causing mutation Deleterious Yes

7 chr1 981434 AGRN 16/36 C T DBSNP 53 0 924 P L Missense Possibly damaging Disease-causing mutation Deleterious Yes7 chr1 7887421 PER3 18/19 C T NOVEL 46 0 803 P L Missense Possibly damaging Benign Deleterious Yes7 chr1 36563558 COL8A2 2/2 G A DBSNP 36 0 575 P L Missense Probably damaging Disease-causing mutation Deleterious7 chr1 52489198 TXNDC12 6/7 G A NOVEL 45 0 135 P L Missense Possibly damaging Disease-causing mutation Deleterious Yes7 chr1 151633333 SNX27 4/12 G T NOVEL 12 0 173 D Y Missense Probably damaging Disease-causing mutation Deleterious7 chr1 237755118 RYR2 32/105 C T NOVEL 36 0 1412 R W Missense Possibly damaging Benign Yes7 chr2 25678304 DTNB 10/18 G A NOVEL 48 0 176 S F Missense Benign Disease-causing mutation Yes7 chr5 13766107 DNAH5 2/21 C T COSMIC 47 0 3360 G E Missense Probably damaging Disease-causing mutation Yes7 chr5 98209411 CHD1 1/10 G A COSMIC 43 0 1153 R * Truncation Disease-causing mutation Yes7 chr6 25653624 SCGN 2/11 G A NOVEL 38 0 33 E K Missense Benign Disease-causing mutation Deleterious Yes7 chr7 938780 ADAP1 11/12 G A NOVEL 62 0 329 T M Missense Probably damaging Disease-causing mutation Deleterious Yes7 chr7 2649749 IQCE 22/22 G A NOVEL 31 0 630 D N Missense Benign Disease-causing mutation Deleterious Yes7 chr8 144940290 EPPK1 1/1 C G DBSNP, COSMIC 18 0 2378 D H Missense Probably damaging Disease-causing mutation7 chr10 108468976 SORCS1 - C T NOVEL 42 0 Splice site Disease-causing mutation7 chr11 15134025 INSC 1/13 C A NOVEL 10 0 4 P T Missense Probably damaging Benign Deleterious7 chr11 66189919 NPAS4 - C T NOVEL 41 0 Splice site Disease-causing mutation7 chr11 92881990 SLC36A4 10/10 G A NOVEL 45 0 275 P S Missense Probably damaging Disease-causing mutation Deleterious Yes7 chr11 102033222 YAP1 3/9 G A NOVEL 32 0 203 R K Missense Benign Disease-causing mutation Deleterious Yes7 chr12 14018971 GRIN2B 2/13 C T NOVEL 43 0 58 D N Missense Probably damaging Disease-causing mutation Tolerated Yes7 chr12 56722350 PAN2 3/26 G A NOVEL 70 0 120 R W Missense Probably damaging Disease-causing mutation Deleterious Yes7 chr12 120784071 MSI1 3/26 C T NOVEL 29 0 218 G E Missense Probably damaging Disease-causing mutation Deleterious Yes7 chr13 23909764 SACS 8/8 C T NOVEL 36 0 2001 E K Missense Probably damaging Disease-causing mutation Tolerated Yes7 chr13 35770072 NBEA 30/57 G A NOVEL 23 0 1667 E K Missense Benign Disease-causing mutation Yes7 chr13 77642818 MYCBP2 12/25 G A NOVEL 36 0 4018 S F Missense Probably damaging Disease-causing mutation Yes7 chr14 78285427 ADCK1 2/9 G A NOVEL 49 0 36 A T Missense Possibly damaging Disease-causing mutation Tolerated Yes7 chr14 105054119 C14orf180 3/5 A G NOVEL 58 0 49 K E Missense Possibly damaging Benign Deleterious7 chr15 72185407 MYO9A 3/19 C T NOVEL 21 0 1752 D N Missense Probably damaging Disease-causing mutation Yes7 chr16 52484232 TOX3 4/7 G A NOVEL 42 0 212 P L Missense Probably damaging Disease-causing mutation Deleterious Yes7 chr19 13409572 CACNA1A 10/38 G A NOVEL 51 0 960 R C Missense Probably damaging Disease-causing mutation Yes7 chr19 40730629 CNTD2 2/5 G A NOVEL 28 0 13 H Splice site Disease-causing mutation7 chr19 51512524 KLK9 2/7 C T NOVEL 60 0 39 G S Missense Probably damaging Disease-causing mutation Deleterious Yes7 chr20 39788792 PLCG1 - C T COSMIC 49 0 Splice site Possibly damaging Disease-causing mutation Yes7 chr22 37906238 CARD10 5/25 A C NOVEL 50 0 297 L W Missense Possibly damaging Disease-causing mutation Deleterious7 chrX 2942083 ARSH 6/9 A C NOVEL 50 0 308 D A Missense Possibly damaging Disease-causing mutation Deleterious Yes7 chrX 117773465 DOCK11 38/53 C T NOVEL 33 0 1357 R * Truncation Disease-causing mutation Yes7 chrX 151366218 GABRA3 8/10 C T COSMIC 48 0 273 R Q Missense Probably damaging Disease-causing mutation Deleterious Yes

9 chr1 115258748 NRAS 2/7 G A COSMIC 6 0 12 G S Missense Benign Disease-causing mutation Deleterious Yes9 chr1 207013267 IL19 4/6 G T NOVEL 9 0 133 E * Truncation Disease-causing mutation9 chr1 215960153 USH2A 52/72 T G COSMIC 44 0 3416 C G Missense Probably damaging Disease-causing mutation9 chr2 63223884 EHBP1 21/25 G A NOVEL 15 0 1100 R H Missense Probably damaging Disease-causing mutation Deleterious Yes9 chr3 175345098 NAALADL2 11/14 A G NOVEL 8 0 607 E G Missense Probably damaging Disease-causing mutation Deleterious9 chr4 88535832 DSPP 4/4 A ATAGCAG NOVEL 42 0 673 D DSSDSSS Inframe insertion Disease-causing mutation9 chr5 88027592 MEF2C 9/12 T A NOVEL 48 0 273 L H Missense Probably damaging Disease-causing mutation Deleterious Yes9 chr6 47647949 GPR111 5/6 C A NOVEL 46 0 205 P H Missense Probably damaging Benign Deleterious Yes9 chr7 45009048 MYO1G 7/17 G T DBSNP 25 0 513 R L Missense Probably damaging Disease-causing mutation Deleterious9 chr8 145541368 DGAT1 11/17 T G NOVEL 20 0 301 V G Missense Probably damaging Disease-causing mutation Deleterious9 chr9 4118418 GLIS3 4/11 G A NOVEL 41 0 354 V M Missense Probably damaging Disease-causing mutation Deleterious Yes9 chr9 72895687 SMC5 6/25 G T NOVEL 7 0 231 E * Truncation Disease-causing mutation9 chr10 117075090 ATRNL1 18/29 T G COSMIC 18 0 961 C G Missense Probably damaging Disease-causing mutation Deleterious9 chr12 23689454 SOX5 14/15 C T COSMIC 49 0 641 R C Missense Probably damaging Disease-causing mutation Deleterious Yes9 chr12 43846379 ADAMTS20 13/39 G A NOVEL 42 0 627 G D Missense Probably damaging Disease-causing mutation Deleterious Yes9 chr13 72049909 DACH1 9/11 C T NOVEL 35 0 650 T M Missense Possibly damaging Disease-causing mutation Yes9 chr14 38723768 CLEC14A 1/1 C T NOVEL 46 0 487 S F Missense Probably damaging Benign Deleterious Yes9 chr15 42968113 STARD9 20/33 A G COSMIC 13 0 605 D G Missense Possibly damaging Benign9 chr15 100801792 ADAMTS17 6/22 G A NOVEL 12 0 308 C Y Missense Probably damaging Disease-causing mutation Deleterious9 chr19 49982165 FLT3LG 1/68 G GC COSMIC 28 0 114-115 Q X Frameshift Disease-causing mutation

21 chr1 104076466 RNPC3 3/15 GA G COSMIC 15 0 116 E X Frameshift Disease-causing mutation21 chr8 144874523 SCRIB 32/37 G A NOVEL 8 0 1461 A T Missense Probably damaging Disease-causing mutation21 chr11 1017183 MUC6 31/33 C A COSMIC 16 0 1873 P Q Missense Probably damaging Benign21 chr12 10588530 KLRC2 1/6 C G COSMIC 29 0 19 R P Missense Probably damaging Benign Deleterious21 chr19 12739215 ZNF791 4/4 A C NOVEL 7 0 291 E A Missense Benign Disease-causing mutation Deleterious

SIFTProtein-damaging prediction

Sanger sequence validation

Targeted sequencing validation

Patient No. Chromosome Hg19 Genome Position

(bp) Gene ExonMutation details Polyphen2

Protein-damaging predictionMutationTaster

Protein-damaging prediction

43 chr1 153233991 LOR 2/2 A ACTCTGG COSMIC 32 0 189 Splice site Disease-causing mutation43 chr2 230456417 DNER 2/13 C A COSMIC 41 0 155 P L Missense Benign Disease-causing mutation Deleterious Yes43 chr4 126329626 FAT4 4/17 C T DBSNP 38 0 1866 T M Missense Probably damaging Disease-causing mutation43 chr5 141039044 ARAP3 - C A NOVEL 39 0 Splice site Disease-causing mutation43 chr5 178416050 GRM6 6/10 G T NOVEL 41 0 414 A T Missense Probably damaging Disease-causing mutation Deleterious Yes43 chr11 108100021 ATM 4/63 T A NOVEL 71 0 101 V D Missense Probably damaging Disease-causing mutation Yes43 chr12 53680313 ESPL1 18/31 C G NOVEL 42 0 1265 R G Missense Possibly damaging Disease-causing mutation Deleterious Yes43 chr13 28602340 FLT3 16/24 C T COSMIC 26 0 676 N K Missense Probably damaging Disease-causing mutation Deleterious Yes43 chr14 93301915 GOLGA5 11/13 C T NOVEL 36 0 653 R * Truncation Disease-causing mutation Yes43 chr14 100604072 EVL - C T DBSNP 34 0 Splice site Disease-causing mutation43 chr17 7577093 TP53 8/11 G C COSMIC 78 0 282 R P Missense Probably damaging Disease-causing mutation Deleterious Yes43 chr17 76082940 TNRC6C 13/21 C T NOVEL 41 0 1187 R C Missense Probably damaging Disease-causing mutation Deleterious Yes43 chr19 42860512 MEGF8 25/41 G A DBSNP 44 0 1443 R H Missense Probably damaging Disease-causing mutation Deleterious Yes43 chr22 43985982 EFCAB6 24/32 G T COSMIC 41 0 1002 E K Missense Possibly damaging Benign Deleterious Yes43 chrX 47836179 ZNF182 7/7 G A NOVEL 9 0 436 G V Missense Probably damaging Disease-causing mutation Deleterious

44 chr1 63877638 ALG6 9/15 C T NOVEL 10 0 241 S F Missense Possibly damaging Disease-causing mutation Deleterious44 chr1 115258748 NRAS 2/7 G T COSMIC 39 0 12 G S Missense Benign Disease-causing mutation Deleterious Yes44 chr2 84934670 DNAH6 54/77 C T NOVEL 13 0 2960 R C Missense Probably damaging Disease-causing mutation Deleterious44 chr3 122545724 DIRC2 3/9 C T DBSNP 49 0 172 T M Missense Benign Disease-causing mutation Deleterious Yes44 chr3 125850318 ALDH1L1 13/23 C A COSMIC 50 0 521 A V Missense Possibly damaging Disease-causing mutation Deleterious Yes44 chr4 147560457-147560466 POU4F2 1/2 TGGCGGC TGGCGGCNOVEL 41 0 55-58 Splice site, indel Disease-causing mutation44 chr5 140769385 PCDHGB4 1/4 G A NOVEL 39 0 645 R H Missense Benign Disease-causing mutation Deleterious44 chr6 16327915-16327918 ATXN1 8/9 ATGC ATGCTGC NOVEL 100 0 208-209 QH QQQH Inframe indel Disease-causing mutation44 chr9 19395661-19395669 HRCT1 1/1 CTCCTTCCCTCCTTCCNOVEL 100 0 100-101 Splice site, indel Disease-causing mutation44 chr12 11214601 TAS2R46 1/1 T G NOVEL 8 0 98 L P Missense Possibly damaging Disease-causing mutation Deleterious44 chr14 92537353 ATXN3 10/11 C CG NOVEL 45 0 315 G AX Frameshift Disease-causing mutation44 chr15 89074487 DET1 3/6 C T NOVEL 8 0 161 D E Missense Possibly damaging Disease-causing mutation Tolerated44 chr19 33490585 RHPN2 10/15 C A DBSNP, COSMIC 17 0 378 Q * Truncation Disease-causing mutation

Supplementary Table 4: A list of mutations in eight iAMP21 patient samples. WES was performed on the matched diagnostic and remission material of eight iAMP21 patient samples (patients 1, 3, 5, 7, 9, 21, 43 and 44). Somatic mutations that were predicted to alter protein function by the majority of protein-damaging predition tools (PolyPhen2, SIFT and Mutation Taster) are listed. DBSNP variants with <1% minor allelic frequency (MAF) were included. Mutations in components of the RAS pathway are highlighted in grey.

1 NRAS Q22K 5% chr1:115258718 C/A Missense Probably damaging Disease-causing mutation Yes1 NF1 P1667S 39% chr17:29653001 C/T Missense Probably damaging Disease-causing mutation No3 KRAS L19F 2% chr12:25398262 G/C Missense Probably damaging Disease-causing mutation Yes3 NRAS E49K 3% chr1:115256566 C/T Missense Benign Disease-causing mutation Yes3 NRAS R68T 3% chr1:115256508 C/G Missense Probably damaging Disease-causing mutation Yes3 PTPN11 E76Q 4% chr12:112888210 G/C Missense Probably damaging Disease-causing mutation Yes4 NRAS G13D 29% chr1:115258744 C/T Missense Benign Disease-causing mutation Yes5 PTPN11 N58S 48% chr12:112888157 A/G Missense Possibly damaging Disease-causing mutation Yes5 PTPN11 A72T 44% chr12:112888198 G/A Missense Probably damaging Disease-causing mutation Yes6 FLT3 L610>WAREYEYDLKWEFPRENL 13% chr13:28608251 51bp insertion Indel NA NA Yes7 FLT3 P606>IWEYDLKWEF 4% chr13:28608239 30bp insertion Indel NA NA Yes8 NRAS R68fs 6% chr1:115256506 GAGA/TTCC Indel NA Disease-causing mutation No8 FLT3 D835V 3% chr13:28592641 T/A Missense Probably damaging Disease-causing mutation Yes9 NRAS G12S 8% chr1:115258748 C/T Missense Possibly damaging Disease-causing mutation Yes9 NRAS G12C 2% chr1:115258748 C/A Missense Possibly damaging Disease-causing mutation Yes9 NRAS G12D 2% chr1:115258747 C/T Missense Benign Disease-causing mutation Yes9 NRAS G13D 5% chr1:115258744 C/T Missense Benign Disease-causing mutation Yes10 KRAS G12D 37% chr12:25398284 C/T Missense Possibly damaging Disease-causing mutation Yes10 KRAS G12D 5% chr12:25398284 C/T Missense Possibly damaging Disease-causing mutation Yes11 FLT3 F605>SRRYEYDLKWEF 9% chr13:28608242 33bp insertion Indel NA NA Yes*12 FLT3 F590fs 9% chr13:28608287 10bp insertion Frameshift NA Disease-causing mutation Yes*12b NRAS G12D 48% chr1:115258747 C/T Missense Benign Disease-causing mutation Yes12b PTPN11 R351Q 48% chr12:112915779 G/A Missense Benign Disease-causing mutation No14 NRAS Q61H 31% chr1:115256528 T/A Missense Benign Disease-causing mutation Yes17 PTPN11 E76K 35% chr12:112888210 G/A Missense Probably damaging Disease-causing mutation Yes18 NRAS Y64_S65insGQEE 42% chr1:115256521 12bp insertion Indel NA Disease-causing mutation No19 NRAS G12S 21% chr1:115258748 C/T Missense Benign Disease-causing mutation Yes19 NRAS G12V 24% chr1:115258747 C/A Missense Possibly damaging Disease-causing mutation Yes20 NRAS G13V 5% chr1:115258744 C/A Missense Probably damaging Disease-causing mutation Yes20 NRAS Y64N 3% chr1:115256521 A/T Missense Probably damaging Disease-causing mutation Yes21 KRAS G12V 11% chr12:25398284 C/A Missense Probably damaging Disease-causing mutation Yes21 FLT3 K602>LADEYFYVDFREYEYDL 13% chr13:28608252 51bp insertion Indel NA NA Yes22 KRAS G12R 26% chr12:25398285 C/G Missense Possibly damaging Disease-causing mutation Yes22 KRAS G12V 10% chr12:25398284 C/A Missense Possibly damaging Disease-causing mutation Yes24 FLT3 I836del 6% chr13:28592637 3bp deletion Indel NA Disease-causing mutation Yes26 FLT3 F612>LDFREYEYDLKWEFPRENLEF 16% chr13:28608220 60bp insertion Indel NA NA Yes*29 NRAS G12S 48% chr1:115258748 C/T Missense Benign Disease-causing mutation Yes30 NRAS G13D 12% chr1:115258744 C/T Missense Benign Disease-causing mutation Yes30 NRAS G13V 6% chr1:115258744 C/A Missense Probably damaging Disease-causing mutation Yes31 KRAS G13D 52% chr12:25398281 C/T Missense Possibly damaging Disease-causing mutation Yes31 KRAS F78L 2% chr12:25380226 A/G Missense Possibly damaging Disease-causing mutation No37 FLT3 N609>RVLSGHVDFREYEYDLKWEFPREN 9% chr13:28608230 69bp insertion Indel NA NA Yes*39 NRAS Q61L 2% chr1:115256529 T/A Missense Probably damaging Disease-causing mutation Yes39 BRAF V600E 44% chr7:140453136 A/T Missense Probably damaging Disease-causing mutation Yes

Supplelmentary Table 5: RAS pathway mutations in iAMP21-ALL. 44 mutations were identified in 26 (25 diagnostic and 1 relapse) iAMP21-ALL patient samples (column A); relapse samples are depicted by 'b', as shown in Supplementary Table 1. Details of the mutation (columns B-D, F), genomic location of the variation (column E) and the predicted functional consequence of the mutation, as predicted by Mutation Taster and Polyphen2 (columns G-I), are shown. Mutations previously identified in cancer are highlighted (column J); yes* defines FLT3- ITD mutations that have been previously reported but the number of inserted amino acids is different.

GenePatient # Nucleotide changeGenome position Reported in COSMICPolyphen2

Protein-damaging MutationTaster

Protein-damaging Type of mutationVariant allele frequency (%)Mutation

- amino acid change

Patient sample Diagnostic lesion Relapse lesion

6BD

RB1 deletion (CN=1) IKZF1 deletion (CN=1)

FLT3 ITD (L610>E611ins18) (VAF, 13%)

IKZF1 deletion (CN=1) CRLF2 rearrangement

12ABC

IKZF1 deletion (CN=1) CRLF2 gain (CN=3)

FLT3 F590_D593fs (VAF, 9%) Ch11:96439395-134944770 (CN=1)

IKZF1 deletion (CN=1) EBF1 gain (CN=3)

CRLF2 rearrangement NRAS G12D (VAF, 48%)

PTPN11 R351Q (VAF, 48%) Ch11:96439395-134944770 (CN=1)

24AD FLT3 I836>M Ch7:120150796-159119708 (CN=1) Ch7:120150796-159119708 (CN=1)

33BC

RB1 deletion (CN=1)

34BC

ETV6 deletion (CN=1)

35BC ETV6 deletion (CN=1)

IKZF1 deletion (CN=1)

36BD RB1 deletion (CN=0) CRLF2 gain (CN=3) RB1 deletion (CN=0)

Supplementary Table 6: Summary of genomic abnormalities in 7 matched diagnostic and relapse iAMP21-ALL samples. Seven diagnostic and relapse matched pairs were interrogated by SNP6.0 array (A) (to identify copy number abnormalities (CNA) of RAS pathway gene loci), MLPA (B) (to identify CNAs of recurrently deleted ALL genes), and targeted sequencing approaches (Screen A (C) and Screen B (D)) (Supplementary Figure 1). All cases were confirmed as iAMP21-ALL at diagnosis and relapse (Supplementary Table 1). The genomic aberrations that were similar at diagnosis and relapse are highlighted in red text. Mutations and CNA identified by MLPA were not detected in the diagnostic sample of patient 34 or the relapse material of patient 33. A single CNA or somatic mutation was not consistent or specific to diagnostic and/or relapse iAMP21-ALL. The genomic profile was frequently different between the 7 matched diagnostic and relapse samples. Abbreviations: VAF, variant allele frequency; CN, gene copy number.